SRM Dilution Calculations 2

Revised for the last time

Today I went to UWPR to make the dilutions. Last night, Laura told me that she diluted our PRTC to 200 fmol/µL, so I had to account for that when making dilutions. The first thing I did was revise my dilution calculations with the new PRTC stock concentration. Because I needed to add more PRTC to each sample, I changed the injection volume from 1 µL to 2 µL for the 10:1 dilution. This means Laura has to pipet 0.375 µL of geoduck, which is less than 0.5 µL. Because Laura and I have pipetted this volume before and it’s only for one sample, I think it is okay. Before I went down, I took 0.1 µL and 0.5 µL pipets from the lab.

Table 1. Revised dilution calculations

Vial

Dilution Ratio

µg to Inject

µL to Inject

µg oyster Needed

µg geoduck Needed

µL oyster needed ((µg/µL)x15 µL/1 µg/µL)

µL geoduck needed ((µg/µL)x15 µL/2 µg/µL)

PRTC added

µL ACN

Total Volume (µL)

1

10:1

1

2

0.9

0.1

6.75

0.375

1.875

6

15

2

7.5:1

1

2

0.87

0.13

6.5

0.5

1.875

6.125

15

3

5:1

1

2

0.8

0.2

6

0.75

1.875

6.35

15

4

2.5:1

1

2

0.6

0.4

4.5

1.5

1.875

7.125

15

5

1:2.5

1

2

0.4

0.6

3

2.25

1.875

7.875

15

6

1:5

1

2

0.2

0.8

1.5

3

1.875

8.625

15

7

1:7.5

1

2

0.13

0.87

1

3.25

1.875

8.875

15

8

1:10

1

1

0.1

0.9

1.5

6.75

3.75

3

15

I then created 25 µL of my pooled oyster solution by adding 5 µL of the samples I selected here.

O14

O39

O96

O124

O137

Using the volumes in Table 1, I prepared the vials by adding my oyster sample, PRTC and ACN. When I got to Vial 6, it was difficult for me to pipet the 1.5 µL I needed, so I made 10 µL of the oyster pool. I finished up the vials and left in the freezer for Laura to add her samples to.